1. Field of the Invention
The present invention relates to recombinant vectors and cell lines, and methods for detecting and monitoring viral infection. More particularly, the invention relates to recombinant vectors and cell lines, and methods for detecting HIV infection, monitoring HIV for drug resistance and screening for an anti-HIV agents.
2. Description of Related Art
Human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerate disease of the immune system termed acquired immune deficiency syndrome (AIDS). Infection of the CD4+ subclass of T-lymphocytes with the HIV type-1 virus (HIV-1) leads to depletion of this essential lymphocyte subclass which inevitably leads to opportunistic infections, neurological disease, neoplastic growth and eventual death.
Infection with human immunodeficiency virus (HIV) is a chronic process with persistent, high rates of viral replication. The pathogenesis of HIV-1 infection is characterized by a variable but often prolonged asymptomic period following the acute viremic phase. Previous work has established a correlation between HIV disease progression and increasing amounts of infectious virus, viral antigens, and virus-specific nucleic acids (Ho et al., New England. J. Med. 321: 1621-1625 (1989); Schnittman et al. AIDS Res. Hum. Retroviruses 7: 361-367 (1991); Pantalco et al. Nature 362: 355-358 (1993)).
A variety of reagents and assays have been developed to detect the infection of HIV and monitor the progression of HIV in the body. For example, counting the depletion of CD4+ cells has been used to indicate the prognosis of AIDS. Serological screening techniques are also being utilized worldwide for the detection of HIV, where the presence of the antibody against HIV antigens, such as the HIV p24 antigen, is detected.
An ELISA assay is currently being utilized on serum samples in most hospitals and screening laboratories to make the determination. However, currently used ELISA assays may not be sensitive enough to detect all HIV infected individuals. This is because that some HIV infected individuals do not have detectable levels of serum antibody to HIV. There may be a significant time lag between detection of HIV infection and seroconversion. In addition, some HIV infected but seronegative individuals might never convert but will remain infected throughout theirs lives. Thus, such a screening method may generate false negatives, which in turn may increases the probability of HIV infection of healthy people by these individuals.
Another method for detecting HIV infection in seronegative individuals was described (Jehuda-Cohen, T. et al. Proc. Natl. Acad. Sci. UAS, 87: 3972-3076 (1990)) wherein peripheral blood mononuclear cells (PBMC) are isolated from the blood and then exposed to a mitogen such as pokeweed mitogen. Incubation of isolated PBMC with pokeweed mitogen caused the PBMC to secret immunoglobulins that were specific for HIV. The failure of the ELISA assay to detect all HIV infected individuals places the population at risk by misleading the HIV infected individuals that they are not infected, thereby making it more likely that the HIV infected individuals will unknowingly infect others.
The existence of HIV has also been determined by using the reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify plasma HIV RNAs (U.S. Pat. No. 5,674,680). This method is used to detect three types of HIV mRNA in peripheral blood cells: unspliced, multiple spliced, and single-spliced mRNA in AIDs patients, HIV-infected but asymptomatic individuals and individuals who are undergoing therapy for AIDS. However, the correlation between the differences in HIV mRNA levels and AIDS prognosis needs to be established.
Many antiviral drugs have been developed to inhibit HIV infection and replication by targeting HIV reverse transcriptase and proteases. Treatment following a prolonged single drug regimen has met with limited success where there is relatively small drop in viral load, followed by a rise in amount of detectable virus in blood, presumably due to the development of drug resistance strains of HIV. The resistance of HIV to drugs is not only associated with the high mutation rates of HIV but also due to the selective pressure of prolonged anti-HIV drug therapy. Since the original description of diminished susceptibility of isolates of HIV-1 to zidovudine (AZT) (Larder et al. Science (1989) 243:1731-1734), the literature has disclosed many descriptions of diminished susceptibility to AZT in different clinical situations, with different assay systems, and of genetic mutations responsible for changes in susceptibility. For example, isolates from subjects not treated with AZT display a narrow range of susceptibilities to AZT, with the 50% inhibitory concentrations (IC50) ranging from 0.001 to 0.04 xcexcM (Larder et al. (1989), supra; Rooke et al. AIDS (1989) 3:411-415; Land et al. J Infect Dis (1990) 161:326-329; Richman et al. J. AIDS (1990) 3:743-746; Tudor-Williams et al. Lancet (1992) 339:15-19). This narrow range of susceptibilities is typical for HIV isolates from subjects of all ages and at all stages of HIV infection. Isolates of HIV from patients who receive AZT, however, chronically display progressive reductions of susceptibility to AZT over periods of months to years. Diminished susceptibility to AZT of an isolate of HIV-2 from a patient on prolonged therapy has also been reported (Pepin et al. Eighth International Conference on AIDS, Amsterdam, The Netherlands, Jul. 19-24, 1992 Abstract PoA 24401).
In addition to AZT, HIV resistance have been seen with other nucleosides and to nonnucleoside anti-retroviral drugs. For example, isolates resistant to AZT display diminished susceptibility to other nucleosides containing a 3xe2x80x2-azido moiety, including 3xe2x80x2-azido-2xe2x80x2, 3xe2x80x2-dideoxyuridine, 3xe2x80x2-azido-2xe2x80x2, xe2x80x2dideoxyguanosine, and 3xe2x80x2-azido-2xe2x80x2,3xe2x80x2-dideoxyadenosine (Larder et al. (1989), supra: Larder et al. Antimicrob Agents Chemother (1990) 34:436-441). Additionally, AZT-resistant isolates are reported to display cross-resistance to didehydrodideoxythymidine (Rooke et al. Antimicrob. Agents Chemother. (1991) 35:988-991).
Drug resistance in HIV isolates is not limited to inhibitors of reverse transcriptase and virtually all drug targets for anti-HIV therapy are susceptible to the development of resistance. For example, a mutant with resistance to a protease inhibitor has been isolated that exhibits an eightfold reduction in susceptibility to a protease inhibitor (Patterson et al. Eighth International Conference on AIDS, Amsterdam, The Netherlands, Jul. 19-24, 1992, Abstract ThA 1506).
In the last five year, with the fast development of anti-HIV drugs and utilization of combination therapy, treatment of HIV infection with multiple antiviral drugs (xe2x80x9ccocktailsxe2x80x9d) have led to diminutions in the amount of viral RNA and virus detectable in blood by using current detection methods. It has been shown that combination therapy with 3 or more antiviral drugs, e.g. indinavir, zidovudine, and lamivudine, or alternatively, nevirapine, zidovudine, and didanosine, in previously untreated patients has resulted in profound decreases in viral burden (Wainberg, M. A. and Friedland, G. JAMA (1998) 279: 1977-1983). It was believed that the combination antiviral regimens used must have blocked viral replication to the extent that the mutations that encode drug resistance could not occur. However, current studies showed that a growing number of patients are failing combination drug regimens (Deek, S. et al. the 5th Conference on Retroviruses and Opportunistic Infection, Chicago, Feb. 1-5, 1998, Abstract #419). Finding an effective salvage therapy for them is difficult.
In the clinical setting, drug resistance is often not detected until a patient manifests symptoms of disease progression, which is generally not observed until significantly after development of a drug resistant strain of virus. Thus, there is a clear need for an assay which can indicate the drug resistance of virus strains so drug therapy for a patient can be modified accordingly, and optimally as soon as resistance is detected rather than delaying until clinical symptoms are observed.
Currently the most commonly used assays for susceptibility of HIV to antiviral drugs involve the measurement of the inhibition of cytopathology, p24 production, or reverse transcriptase production of a laboratory strain of HIV in a lymphoblastoid cell line. Such assays may not be readily applied to clinical isolates of HIV. Examples of commonly used assays of drug susceptibility of clinical isolates have been the syncytial focus assay in CD4-HeLa cells (Chesebro, B. and Wehrly, K., J. Virol. (1988) 62:3779-3788), inhibition of p24 production in primary peripheral blood mononuclear cells, and reverse transcriptase (RT) assays using cultured primary T-cells from patient blood. (Richman et al. In:Current Protocols in Immunology, Coligan et al., eds, (1993) Brooklyn, J. Wiley).
One of the disadvantages associated with the syncytial focus assay is that it may only detect HIV viruses that exhibit a syncytial-inducing phenotype and that in practice may only be obtained from a minority of specimens from seropositive individuals. And the syncytial focus assays may not be used for screening for drugs that affect posttranslational processing, such as glycosidase and protease inhibitors. On the other hand, the p24 and RT assays may also suffer the limitations of difficult quantitation, low sensitivity and unproven clinical validity.
A recombinant cell is provided which comprises:a reporter sequence introduced into the recombinant cell comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; the recombinant cell being capable of cell division and expressing a CD4 receptor and one or more additional cell surface receptors which facilitate productive infection of the recombinant cell by the HIV virus; and the recombinant cell enabling HIV virus which has infected the recombinant cell to replicate and infect non-infected cells in a culture of the recombinant cell.
As used herein, introducing a reporter sequence into a recombinant cell refers to the introduction of a sequence into cell by any of a variety of recombinant methodologies including, but not limited to, transformation, transfection and transduction.
The recombinant cell may optionally express a sufficient number of cell surface receptors to render the recombinant cell permissive to substantially all strains of HIV. Alternatively, the recombinant cell may express a selected group of cell surface receptors such that the recombinant cell is permissive to a selected group of strains of HIV. Examples of cell surface receptors which may be expressed by the recombinant cell include, but are not limited to CXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3, CX3CR1, STRL33/BONZO and GPR15/BOB.
The stably transferred reporter sequence may optionally comprise a promoter sequence including an HIV virus specific enhancer sequence, and a reporter gene whose expression is regulated by binding of an HIV specific transactivator protein to the HIV specific enhancer sequence. According to this variation, the HIV specific transactivator protein is preferably Tat and the HIV specific enhancer sequence preferably comprises at least one copy of TAR sequence. Alternatively, the HIV specific protein may optionally regulates expression of the reporter sequence by a protein-protein interaction between the HIV specific protein and a transactivator protein present in the recombinant cell.
Examples of the HIV specific protein include, but are not limited to, HIV proteins Tat, Rev, Vpr, Vpx, Vif, Vpu, Nef, Gag, Env, RT, PR, and IN. The HIV specific protein may optionally be an HIV transactivator protein such as Tat.
Expression of the reporter gene in the recombinant cell may be is up-regulated or down-regulated by the HIV specific protein.
A method is provided for detecting a presence of HIV virus in a sample comprising: taking a culture of recombinant cells which (a) are capable of cell division, (b) express CD4 receptor and one or more additional cell surface receptors necessary to allow the HIV virus to infect, (c) enable the HIV virus to replicate and infect the noninfected cells in the cell culture, and (d) comprise a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; contacting the cell culture with a sample to be analyzed for the presence of HIV virus in the sample; and detecting a change in a level of expression of the reporter gene in cells in the recombinant cell culture.
A method is also provided for detecting the presence of different strains of HIV virus in a sample comprising: taking a first culture of recombinant cells which (a) are capable of cell division, (b) express CD4 receptor and one or more additional cell surface receptors which render the first cell culture permissive to a first group of strains of HIV but does not render the first cell culture permissive to a second, different group of strains of HIV, (c) enable the HIV virus to replicate and infect the noninfected cells in the cell culture, and (d) comprise a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; taking a second culture of recombinant cells which (a) are capable of cell division, (b) express CD4 receptor and one or more additional cell surface receptors which render the second culture permissive to the second group of strains of HIV but does not render the second cell culture permissive to the first group of strains of HIV, (c) enable the HIV virus to replicate and infect the noninfected cells in the cell culture, and (d) comprise a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; contacting the first and second cell cultures with a sample to be analyzed for the presence of different strains of HIV virus; detecting a change in a level of expression of the reporter gene in cells in the first cell culture; detecting a change in a level of expression of the reporter gene in cells in the second cell culture; and distinguishing between the first and second groups of strains based on whether a change in a level of expression of the reporter gene occurs in the first or the second cell culture.
According to the above method, the first and second cultures of recombinant cells may optionally be mixed with each other. The reporter genes in the first and second cultures of recombinant cells may also optionally be different from each other so that cells of the first cell culture can be distinguished from cells of the second cell culture. This allows different strains of HIV virus to be detected in a single well containing cells from both cultures.
A method is also provided for detecting HIV drug resistance in a sample comprising: taking a culture of recombinant cells which (a) are capable of cell division, (b) express CD4 receptor and one or more additional cell surface receptors necessary to allow the HIV virus to infect, (c) enable the HIV virus to replicate and infect the noninfected cells in the cell culture, and (d) comprise a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; contacting the cell culture with a sample containing HIV virus; adding one or more anti-HIV agents to the cell culture either before or after contacting the cell culture with the sample; and detecting a change in a level of expression of the reporter gene in the cells.
A method is also provided for taking a patient known to be infected with one or more strains of the HIV virus and determining what combination of one or more anti-HIV agents would be effective in treating the patient, the method comprising: taking a plurality of cell cultures, each of the cultures containing recombinant cells which (a) are capable of cell division, (b) express CD4 receptor and one or more additional cell surface receptors necessary to allow the HIV virus to infect, (c) enable the HIV virus to replicate and infect the noninfected cells in the cell culture, and (d) comprise a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; contacting the cell cultures with a sample containing the HIV virus; adding a different set of one or more anti-HIV agents to each of the cell cultures, either before or after contacting the cell cultures with the sample; and comparing expression of the reporter gene in the plurality of cell cultures.
A method for screening compositions for anti-HIV activity comprising: taking a culture of recombinant cells which (a) are capable of cell division, (b) express CD4 receptor and one or more additional cell surface receptors necessary to allow the HIV virus to infect, (c) enable the HIV virus to replicate and infect the noninfected cells in the cell culture, and (d) comprise a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; contacting the cell culture with a sample containing the HIV virus; adding one or more agents whose anti-HIV activity are unknown to the cell culture, either before or after contacting the cell cultures with the sample; and detecting a change in a level of expression of the reporter gene in the cells in the culture.
According to any one of the above methods, the recombinant cells in the cell cultures used in the methods may optionally comprise a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; the recombinant cells being capable of cell division and expressing a CD4 receptor and one or more additional cell surface receptors which facilitate productive infection of the recombinant cell by the HIV virus; and the recombinant cells enabling the HIV virus which has infected the recombinant cell to replicate and infect non-infected cells in a culture of the recombinant cell.
Also according to any one of the above methods, the HIV specific protein may be any one of the HIV proteins Tat, Rev, Vpr, Vpx, Vif, Vpu, Nef, Gag, Env, RT, PR, and IN. The HIV specific protein may optionally be an HIV transactivator protein such as Tat.
Also according to any one of the above methods, the reporter sequence may comprise a promoter sequence including an HIV virus specific enhancer sequence, and a reporter gene whose expression is regulated by binding of an HIV specific transactivator protein to the HIV specific enhancer sequence. In one variation, the HIV specific transactivator protein is Tat and the HIV specific enhancer sequence comprises at least one copy of TAR sequence.
Also according to any one of the above methods, the one or more additional cell surface receptors expressed by the recombinant cell may include, but are not limited to CXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3, CX3CR1, STRL33/BONZO and GPR15/BOB.
Also according to any one of the above methods, detecting a change in a level of expression of the reporter gene in the cells may include detecting a change in a level of expression of the reporter gene in individual cells.
Also according to any one of the above methods, detecting a change in a level of expression of the reporter gene in the cells may include detecting a change in a level of expression of the reporter gene across the cell culture.
Also according to any one of the above methods, detecting a change in a level of expression of the reporter gene in the cells may include detecting whether viral replication within the cell culture has occurred.
Also according to any one of the above methods, detecting a change in a level of expression of the reporter gene in the cells may include comparing a level of expression in cells contacted with the sample to a level of expression cells contacted with one or more control samples.
Also according to any one of the above methods, the sample may be any sample which might include HIV including, but not limited to whole blood, blood serum, isolated peripheral blood cells,T cells, and bone marrow.
Kits are also provided for performing the various methods of the present invention. These kits may include the cell line of the present invention and any two or more components used to perform these methods.
In one variation, a kit is provided which comprises: first and second recombinant cell lines, each recombinant cell line comprising: a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus, the recombinant cell line being capable of cell division and expressing a CD4 receptor and one or more additional cell surface receptors which facilitate productive infection of the recombinant cell by the HIV virus, and the recombinant cell line enabling the HIV virus which has infected the recombinant cell to replicate and infect non-infected cells in a culture of the recombinant cell; wherein the one or more additional cell surface receptors which the first recombinant cell line expresses renders the first recombinant cell line permissive to a first group of strains of HIV and the one or more additional cell surface receptors which the second recombinant cell line expresses renders the second recombinant cell line permissive to a second, different group of strains of HIV.
According to this variation, the first and second recombinant cell lines may optionally be mixed together in the kit. Also according to this variation, the first recombinant cell line may optionally include a first reporter gene and the second recombinant cell line may optionally include a second different reporter gene which allows the first and second recombinant cell lines to be independently identified.
The present invention also provides a recombinant viral vector for producing the recombinant cell described above. The recombinant viral vector comprises: a reporter sequence comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of a cell transduced by the recombinant viral vector; and a receptor sequence comprising a CD4 gene and one or more coreceptor genes, expression of the receptor and coreceptor genes facilitating productive infection of the transduced cell and enabling HIV virus which has infected the transduced cell to replicate and infect non-infected cells in a culture of the cells transduced by the recombinant viral vector.
In a preferred embodiment, the recombinant viral vector is a recombinant adenoviral vector. The recombinant adenoviral vector may be replication incompetent but carry an adenoviral packaging signal. The vector carries genes encoding HIV receptors, such as CD4, CXCR4 and CCR5, as well as a reporter gene such as xcex2-galactosidase, luciferase, beta-glucuronidase, fluorescent protein (e.g. GFP and BFP), chloramphenicol acetyl transferase (CAT), secreted embryonic alkaline phosphatase (SEAP), hormones and cytokines. The vector may also carry a gene encoding an interleukin (e.g. IL-2 and IL-12) that renders the transduced cells more susceptible to HIV infection. The vector may also carry a eukaryotic polyadenylation sequence such a SV40 polyadenylation site or a BGH polyadenylation site.
The genes encoding the HIV receptors may be placed under transcriptional control of a constitutive (e.g. CMV and SV40) or an inducible (e.g. tetracycline-inducible) promoter located in the E1 region of the adenoviral vector near the left terminal repeats (L-TR). The reporter sequence may be positioned in the right end of the recombinant adenoviral vector, for example, in the E4 region of the recombinant adenoviral vector near the right terminal repeats (R-TR).
Various HIV receptors may be transferred into the cells by a single recombinant viral vector carrying all of the HIV receptors, or by multiple recombinant viral vectors, each carrying one or more HIV receptors to confer upon the cell different tropisms.
Alternatively, a recombinant plasmid may be used to introduce the receptor and reporter sequences to the cell. The recombinant plasmid comprises: a reporter sequence comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of a cell transfected with the recombinant plasmid; and a receptor sequence comprising a CD4 gene and one or more coreceptor genes, expression of the receptor and coreceptor genes facilitating productive infection of the transfected cell and enabling HIV virus which has infected the transfected cell to replicate and infect non-infected cells in a culture of the cells transfected with the recombinant plasmid.
The present invention also provides a kit for producing the recombinant cells described above. The kit comprises: a recombinant viral vector and a cell line capable of being infected by the vector, the recombinant viral vector comprising a reporter sequence comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of a cell in the cell line that is transduced by the recombinant viral vector, and a receptor sequence comprising a CD4 gene and one or more coreceptor genes, expression of the receptor and coreceptor genes facilitating productive infection of the transduced cell and enabling HIV virus which has infected the transduced cell to replicate and infect non-infected cells in a culture of the cells transduced by the recombinant viral vector.
The present invention also provides a method for producing recombinant cells for detecting a presence of HIV virus in a sample. The method comprises: taking a culture of cells; and adding a recombinant viral vector into the culture to transduce the cells, the recombinant viral vector comprising a reporter sequence comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of a cell in the culture that is transduced by the recombinant viral vector, and a receptor sequence comprising a CD4 gene and one or more coreceptor genes, expression of the receptor and coreceptor genes facilitating productive infection of the transduced cell and enabling HIV virus which has infected the transduced cell to replicate and infect non-infected cells in the culture of the cells transduced by the recombinant viral vector.
Alternatively, the recombinant cells of the present invention may be produced by transducing cells that already express CD4 and one or more HIV coreceptors such as CXCR4 and CCR5 with a recombinant viral vector containing the reporter sequence.
Optionally, the recombinant cells of the present invention may also be produced by transducing cells that already contain the reporter sequence with a recombinant viral vector that expresses CD4 and one or more HIV coreceptors.
Optionally, the recombinant cells of the present invention may also be produced by transducing cells that already express CD4 and one or more HIV coreceptors such as CXCR4 and CCR5 at levels sufficient for facilitating productive infection of HIV virus in the cells with a recombinant viral vector containing the reporter sequence.
The recombinant cells of the present invention may also be produced by transducing cells with a plurality of recombinant viral vectors, each of the it plurality of recombinant viral vectors expressing the receptor sequence such as genes encoding CD4, CXCR4 and CCR5, or the reporter sequence.
The recombinant viral vector of the present invention may also be used to transduce cells that express CD4 or a coreceptor (e.g. CXCR4 and CCR5) naturally, but at lower levels than the expression levels conferred by an artificial expression system, such as those provided by the recombinant expression vectors of the present systems. By introducing a vector carrying the HIV receptor(s) into the cell, the expression levels of the HIV receptor(s) may be significantly elevated by using strong promoters (such as CMV and SV40 promoters) to overexpress the receptor(s).
The recombinant viral vector of the present invention may also be used to produce cells that express the receptors in a controlled period of time by using an inducible promoter, or in a shorter period of time by using an adenoviral vector. This allows versatile and efficient production of a wide variety of cells which can be used for detecting HIV infection in the cell, screening for anti-HIV drugs and detecting HIV drug resistance in the cells.